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Characterizing the interaction between TTP and the 4EHP- GYF2 complex


Precise control of gene expression involves multiple steps beyond transcription, including mRNA turnover and translational regulation. One pathway involves the RNA- binding protein tristetraprolin (TTP), which binds to AU- rich elements (AREs) in the 3' UTR of certain cytokine mRNAs and promotes their decay by recruiting degradation factors. Though TTP's role in mRNA decay is well established, its role in translational repression is poorly understood. Recently, the Lykke-Andersen lab, by mass spectrometry in collaboration with the Bennett Lab, revealed a novel interaction between TTP and the translational repressor complex 4EHP-GYF2. Aside from the potential insight into the mechanism of TTP-mediated translational repression, this finding also suggests TTP- mediated mRNA decay and translational repression could be interdependent. Bacterially expressed recombinant TTP, 4EHP, and GYF2 in in vitro pull-down experiments revealed weak binding of 4EHP to TTP, but strong enrichment for binding upon the addition of GYF2, indicating GYF2 is important in bridging this complex together. The better understand the molecular interaction, the domains of TTP needed for binding GYF2 and 4EHP were mapped out by mutagenesis. Co-immunoprecipitation of wild-type (WT) and deletion mutants of TTP expressed in HEK293T cells revealed the N-terminal domain of TTP to be necessary and sufficient for binding to the 4EHP-GYF2 complex and further mapping revealed conserved PPPPGF motifs, that when mutated, results in almost complete loss of binding to the 4EHP-GYF2 complex. The functional implication of these interactions was tested in in vitro translation experiments; however, only modest translational repression by the TTP-4EHP-GYF2 complex was observed under those conditions

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